Figure 2
From: Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression
Significant role for EPACs in α-MSH-induced CRE activation: GT1-7 cells.
(A) cAMP accumulation was measured after labeling of GT1-7 cells with [3H]-adenine followed by the purification of [3H]cAMP and [3H]ATP by sequential chromatography. Cells were stimulated with various concentrations of α-MSH and γ-MSH for 30 min at 37 °C. Means of 5 independent experiments performed in quadruplicates were compiled. Asterisks indicate a significant difference to cells stimulated either with the lowest concentration of α-MSH (−11) or γ-MSH (−9) using one-way ANOVA followed by Dunnett’s analysis. (B) GT1-7 cells were transiently transfected with a reporter gene construct harbouring the firefly luciferase gene under control of a CRE-dependent promoter. Cells were stimulated or not with 1 μM of α-MSH for 4 h after 30 min pretreatment with KT-5720 (5 μM; N = 7), A-812511 (10 μM; N = 4), rp-Br-cAMPs (50 μM; N = 5), ESI-09 (20 μM; N = 5), HJC-0197 (25 μM; N = 3) or ESI-05 (50 μM; N = 5) or the carrier DMSO (0.1% or 0.2%). Data were compiled first by normalizing α-MSH-induced CRE activation to the carrier or to the inhibitor alone and then by calculating the inhibition as percentage. Hashed signs indicate a significant difference to zero using the one-sample Student’s t-test.
